Lipidated peptides as neuroprotective agents

ABSTRACT

Lipidated neuropeptides based on PrRP31, PrRP20, containing C14 and/or C16 fatty acid, in which sequence of IRPVGRF-NH 2  at the C-terminus is variable in the site of isoleucine, valine and phenylalanine; the fatty acid is bound in position 1 or 11 for PrRP31 or its analog and in position 1 or 7 for PrRP20 or its analog; the fatty acid is bound directly or through a hydrophilic linker X 2 , for use in the treatment and prevention of neurodegenerative diseases such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.

FIELD OF THE INVENTION

New analogs of prolactin releasing peptide (PrRP) represent neuroprotective agents for peripheral treatment and prevention of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.

BACKGROUND OF THE INVENTION

AD is a serious neurodegenerative brain disease affecting mainly older people. The disease starts to manifest with memory decline, learning disorders, behavioral changes, impairment in orientation in time and space, loss of autonomic functions, finally results in complete dementia. The death comes on average 9 years after diagnosis. Histopathologically, AD is characterized by two hallmarks: intracelullar neurofibrilary tangles formed by hyperphosphorylated Tau protein and extracellular senile plaques of beta peptide.

Prolactin releasing peptide (PrRP) was discovered at the end of 20^(th) century. Naturally two isoforms of PrRP can be found in organism: peptide containing 31 amino acids (PrRP/1-31/; PrRP31) or 20 amino acids (PrRP/12-311; PrRP20), its amino acid composition also exhibits small differences in various species (human, rat, bovine) (Hinuma et al., 1998). PrRP is produced in neurons of many brain regions, mainly in medulla oblongata (in nucleus tractus solitarius and ventrolateral reticular nucleus), and hypothalamus (in paraventricular and dorsomedial nuclei), less in pituitary gland, and amygdala. In the periphery PrRP can be found in adrenal medulla, testis, pancreas, and small and large intestines.

PrRP receptor, GPR10, is extensively expressed in the whole brain; it can be found in anterior pituitary, amygdala, hypothalamus, brainstem, and medulla oblongata. In the periphery GPR10 can be found in adrenal medulla, and significantly increased expression was observed in human and rat pancreas.

Subsequently, new modified analogs of neuropeptides PrRP31 and PrRP20 were synthesized, with changes in amino acid chain, lipidated with fatty acid (e.g. myristoylated or palmitoylated) at the N-terminus, however, for use in regulating food intake (WO2014/009808) and regulating blood glucose levels (U.S. Pat. No. 61/927944).

Nowadays, drugs slowing the AD progression and improving cognitive functions are used. These are inhibitors of acetylcholinesterase, which increase acetylcholine concentration in the brain and inhibitors of N-methyl-D-aspartate receptors (e.g. memantin). Because of the high incidence of insulin resistance in AD patients, it is not possible to use insulin as an AD treatment. It is hypothesized that agents increasing insulin sensitivity, such as metformin, insulin secretagogues such as glucagon-like peptide-1 (GLP-1 gastric-inhibitory peptide (GIP and their analogs could act as AD treatment.

There is a need to provide further substances with neuroprotective effect which could be useful in the treatment of neurodegeneravie diseases.

DISCLOSURE OF THE INVENTION

The present invention provides lipidated neuropeptides based on prolactin-releasing peptide (PrRP-based neuropeptides) selected from prolactin-releasing peptide 20 (PrRP20), prolactin-releasing peptide 31 (PrRP31) and their analogs, wherein in the C-terminal sequence IRPVGRF-NH₂ (SEQ ID NO. 1), one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; said PrRP-based neuropeptide containing C14 and/or C16 fatty acid chain, said fatty acid is bound in position 1 or 11 for PrRP31 or its analogs and in position 1 or 7 for PrRP20 or its analogs; said fatty acid being bound by a bond between an amino acid having at least one free NH₂, OH or SH group and the carboxylic group of the fatty acid or through a hydrophilic linker X² selected from the group comprising polyoxyethylene moiety, arylalkyl moiety, or a saturated or unsaturated, linear or branched C₃-C₈ hydrocarbon chain, wherein some carbon atoms may be replaced by heteroatoms selected from a group comprising N, S, and O; said chain carrying at least one and preferably two amino groups or carboxylic acid groups, one of which may be substituted to form a group selected from: CONH₂; NH-polyoxyethylene; COOM¹ wherein M¹ is alkali metal, preferably Na or K; CN; COOR¹, COR¹, or CONHR¹ wherein R¹ is selected from a group comprising lower alkyl, arylalkyl, polyoxyethylene, methylpolyoxyethylene, and aminoethylpolyoxyethylene; (CHOH)_(n)R² where R² is H or COOH and n is an integer from 2 to 10; or (CH)_(n)N⁺R₃, wherein R₃ is the same or different, selected from H and C₁-C₄ alkyl;

and the PrRP31 or its analogs may optionally have the amino acid in position 11 replaced by an amino acid having a free NH₂, OH or SH group, particularly when the fatty acid is bound in position 11 for PrRP31 or its analogs; and the PrRP20 or its analogs may have the amino acid in position 7 replaced by an amino acid having a free NH₂, OH or SH group in position 7, particularly when the fatty acid is bound in position 7 for PrRP20 or its analogs; for use in a method of treatment and prevention of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.

The PrRP31 and/or PrRP20 include variants found in various animal species. Preferred are the human and rat variants.

Preferably, in the sequence of the C-terminal heptapeptide as mentioned above, isoleucine can be replaced by phenylglycine or alanine, valine can be replaced by phenylglycine and/or terminal phenylalanine can be replaced by dichlorophenylalanine, pentafluorophenylalanine, nitrophenylalanine, histidine, benzylhistidine, naphthylalanine, tryptofane, pyroglutamic acid, benzylcysteine, benzyl-O-glutamate, tetrachlorophenylalanine, methyl-O-phenylalanine or methyl-NH-phenylalanine.

The binding of the fatty acid thus includes either a direct bond between an amino acid of the PrRP chain having at least one free amino, SH or OH group and the carboxylic group of the fatty acid, or a bond through X², wherein X² is a hydrophilic linker selected from a group comprising polyoxyethylene moiety, arylalkyl moiety, or a saturated or unsaturated, linear or branched C₃-C₈ hydrocarbon chain, wherein some carbon atoms may be replaced by heteroatoms selected from a group comprising N, S, and O; said chain carrying at least one and preferably two amino groups or carboxylic acid groups, one of which may be substituted to form a group selected from: CONH₂; NH-polyoxyethylene; COOM¹ wherein M¹ is alkali metal, preferably Na or K; CN; COOR¹, COR¹, or CONHR¹ wherein R¹ is selected from a group comprising lower alkyl, arylalkyl, polyoxyethylene, methylpolyoxyethylene, and aminoethylpolyoxyethylene; (CHOH)_(n)R² wherein R² is H or COOH and n is an integer from 2 to 10; or (CH)_(n)N⁺R₃, where R₃ is the same or different, selected from H and C₁-C₄ alkyl.

Preferably, X² is a hydrophilic linker selected from the group comprising β-alanine, γ-aminobutyric acid and γ-glutamic acid.

When the fatty acid is bound in position 11 for PrRP31 or its analogs, the PrRP31 or its analogs have an amino acid having a free NH₂, OH or SH group in position 11, and when the fatty acid is bound in position 7 for PrRP20 or its analogs, the PrRP20 or its analogs have an amino acid having a free NH₂, OH or SH group in position 7. Amino acids having a free NH₂, OH or SH group include, for example, lysine, arginine, serine, cysteine, tyrosine.

The present invention provides, more particularly, the lipidated analogs of PrRP20 or PrRP31 (rat and human) according to the formulae:

(1) (SEQ ID NO. 2) (X)SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH₂, (2) (SEQ ID NO. 3) (X)SRAHQHSMETRTPDINPAWYTGRGIRPVGRF-NH₂,  or (3) (SEQ ID NO. 4) (X)TPDINPAWYASRGIRPVGRF-NH₂, (4) (SEQ ID NO. 5) (X)TPD1NPAWYTGRGIRPVGRF-NH₂, wherein X═X¹ or X¹X²; X¹ being tetradecanoic or hexadecanoic acid, which is bound in a position 1 to an amino acid of the above mentioned peptide chain either directly or through X², X² being a hydrophilic linker as defined above, preferably selected from the group consisting of β-alanine, γ-amino butyric acid and γ-glutamic acid, and wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; for use in the treatment and prevention, preferably by peripheral administration, of neurodegenerative diseases, which are Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.

In a preferred embodiment, the lipidated analogs of PrRP20 or PrRP31 according to the formulae:

(5) (SEQ ID NO. 6) (N-palm)SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH₂ and (6) (SEQ ID NO. 7) (palm)TPDINPKWYASRGIRPVGRF-NH₂; wherein palm is hexadecanoic acid, and wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; are provided for use in the treatment and prevention, preferably by peripheral administration, of neurodegenerative diseases, which are Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.

In another embodiment, the lipidated analogs of PrRP20 or PrRP31 according to the formulae:

(7) (SEQ ID NO. 8) SRTHRHSMEIK(palm)TPDINPAWYASRGIRPVGRF-NH₂, (8) (SEQ ID NO. 9) TPDINPK(palm)WYASRGIRPVGRF-NH₂, (9) (SEQ ID NO. 10) SRTHRHSMEIKTPDINPAWYASRGIRPVGRF-NH₂,  and           |          X²(palm) (10) (SEQ ID NO. 11) TPDINPKWYASRGIRPVGRF-NH₂;       |      X²(palm) wherein palm is hexadecanoic acid and X² is γ-glutamic acid, and wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; are provided for use in the treatment and prevention, preferably by peripheral administration, of neurodegenerative diseases, which are Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.

A further embodiment of the invention relates to the use of lipidated neuropeptides based on prolactin-releasing peptide (PrRP-based neuropeptides) selected from prolactin-releasing peptide 20 (PrRP20), prolactin-releasing peptide 31 (PrRP31) and their analogs, wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; said PrRP-based neuropeptide containing C14 and/or C16 fatty acid chain, said fatty acid is bound in position 1 or 11 for PrRP31 or its analogs and in position 1 or 7 for PrRP20 or its analogs; said fatty acid being bound by a bond between an amino acid having at least one free NH₂, OH or SH group and the carboxylic group of the fatty acid or through a hydrophilic linker X² selected from the group comprising polyoxyethylene moiety, arylalkyl moiety, or a saturated or unsaturated, linear or branched C₃-C₈ hydrocarbon chain, wherein some carbon atoms may be replaced by heteroatoms selected from a group comprising N, S, and 0; said chain carrying at least one and preferably two amino groups or carboxylic acid groups, one of which may be substituted to form a group selected from: CONH₂; NH-polyoxyethylene; COOM¹ wherein M¹ is alkali metal, preferably Na or K; CN; COOR¹, COR¹, or CONHR¹ wherein R¹ is selected from a group comprising lower alkyl, arylalkyl, polyoxyethylene, methylpolyoxyethylene, and aminoethylpolyoxyethylene; (CHOH)_(P)R² where R² is H or COOH and n is an integer from 2 to 10; or (CH)_(n)N⁺R₃, where R₃ is the same or different, selected from H and C₁-C₄ alkyl;

and the PrRP31 or its analogs may have the amino acid in position 11 replaced by an amino acid having a free NH₂, OH or SH group, particularly when the fatty acid is bound in position 11 for PrRP31 or its analogs; and the PrRP20 or its analogs may have the amino acid in position 7 replaced by an amino acid having a free NH₂, OH or SH group in position 7, particularly when the fatty acid is bound in position 7 for PrRP20 or its analogs; for the manufacture of a medicament for treatment and prevention of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.

Another embodiment of the invention provides a method of treatment and prevention of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders, comprising a step of administering to a subject in need of such treatment or prevention, preferably by peripheral administration, lipidated neuropeptides based on prolactin-releasing peptide (PrRP-based neuropeptides) selected from prolactin-releasing peptide 20 (PrRP20), prolactin-releasing peptide 31 (PrRP31) and their analogs, wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; said PrRP-based neuropeptide containing C14 and/or C16 fatty acid chain, said fatty acid is bound in position 1 or 11 for PrRP31 or its analogs and in position 1 or 7 for PrRP20 or its analogs; said fatty acid being bound by a bond between an amino acid having at least one free NH₂, OH or SH group and the carboxylic group of the fatty acid or through a hydrophilic linker X² selected from the group comprising polyoxyethylene moiety, arylalkyl moiety, or a saturated or unsaturated, linear or branched C₃-C₈ hydrocarbon chain, wherein some carbon atoms may be replaced by heteroatoms selected from a group comprising N, S, and O; said chain carrying at least one and preferably two amino groups or carboxylic acid groups, one of which may be substituted to form a group selected from: CONH₂; NH-polyoxyethylene; COOM¹ wherein M¹ is alkali metal, preferably Na or K; CN; COOR¹, COR¹, or CONHR¹ wherein R¹ is selected from a group comprising lower alkyl, arylalkyl, polyoxyethylene, methylpolyoxyethylene, and aminoethylpolyoxyethylene; (CHOH)_(n)R² wherein R² is H or COOH and n is an integer from 2 to 10; or (CH)_(n)N⁺R₃, where R₃ is the same or different, selected from H and C₁-C₄ alkyl;

and the PrRP31 or its analogs may have the amino acid in position 11 replaced by an amino acid having a free NH₂, OH or SH group, particularly when the fatty acid is bound in position 11 for PrRP31 or its analogs; and the PrRP20 or its analogs may have the amino acid in position 7 replaced by an amino acid having a free NH₂, OH or SH group in position 7, particularly when the fatty acid is bound in position 7 for PrRP20 or its analogs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the escape latency in Morris water maze test (MWM). The experiment was performed for 5 days, with 4 sessions per day, each session from different starting point, using MSG obese mice and their controls. Data are mean±SEM, n=10 mice per group.

Statistical analysis is 2-way ANOVA with Bonferroni post hoc test. Significance is *P <0.05 and **P<0.01.

FIG. 2 shows phosphorylation of GSK-3β at Ser9 and Tau phosphorylation at Ser396 and Thr231 in the hippocampi of 2 and 6-month-old MSG mice and their age-matched controls. Phosphorylation was determined using Western blot (WB). Data are mean±SEM, n=7-10 mice per group. Statistical analysis is 1-way ANOVA with Bonferroni post hoc test. Significance is *P<0.05 and ***P<0.001.

FIG. 3 shows insulin signaling cascade in hippocampi of 6-month-old MSG obese mice after 14-day treatment with liraglutide and palmitoylated human PrRP31. Liraglutide (0.2 mg/kg) and palmitoylated human PrRP31 (5 mg/kg) were subcutaneously administered twice a day, in the morning and in the evening. Saline treated mice served as a control. Phosphorylation was determined using WB. Data are mean±SEM, n=7-10 mice per group. Statistical analysis is 1-way ANOVA with Bonferroni post hoc test. Significance is *P<0.05 and ***P<0.001.

FIG. 4 shows phosphorylation at different epitopes of Tau protein in hippocampi of 6-month-old MSG mice after 14-day treatment with liraglutide and palmitoylated human PrRP31. Liraglutide (0.2 mg/kg) and palmitoylated human PrRP31 (5 mg/kg) were subcutaneously administered twice a day, in the morning and in the evening. Saline treated mice served as a control. Phosphorylation was determined using WB. Data are mean SEM, n=7-10 mice per group. Statistical analysis is 1-way ANOVA with Bonferroni post hoc test. Significance is *P<0.05 and ***P<0.001.

FIG. 5 shows phosphorylation of Tau protein at different Tau epitopes in hippocampi of 6-month-old MSG mice after 14-day treatment with palmitoylated PrRP31 with dichlorophenylalanin in position 31. Palmitoylated PrRP31 with dichlorophenylalanin in position 31 (5 mg/kg) was subcutaneously administered twice a day, in the morning and in the evening. Saline treated mice served as a control. Phosphorylation was determined using WB. Data are mean±SEM, n=7-10 mice per group. Statistical analysis is 1-way ANOVA with Bonferroni post hoc test. Significance is *P<0.05 and ***P<0.001.

FIG. 6 shows immunohistochemical analysis of Tau hyperphosphorylation in CA1 region of the hippocampus of 6-month-old MSG mice and their age-matched controls, and Tau hyperphosphorylation after 14-day treatment with liraglutide and palmitoylated PrRP31. Liraglutide (0.2 mg/kg) and palmitoylated human PrRP31 (5 mg/kg) were subcutaneously administered twice a day, in the morning and in the evening. Saline treated mice served as a control. Tau phosphorylation was determined using double immunohistochemical fluorescent staining.

FIG. 7 shows spatial memory testing of Thy-Tau 22 mice and their WT controls in Y-maze. The memory was tested A/ before the beginning of the experiment and B/ after 2-month-long treatment with LiPR31 dissolved in PBS/5% Tween 80 using SC Alzet® osmotic pumps, the concentration was 5 mg/kg/day; PBS/5% Tween 80 was administered to the control group. Data are mean±SEM, n=10-12 animals per group. Statistical analysis is Student t-test. Significance is *P<0.05.

FIG. 8 shows Tau phosphorylation after 2-month-long treatment of Thy-Tau22 mice with LiPR31 dissolved in PBS/5% Tween 80 using SC Alzet® osmotic pumps, the concentration was 5 mg/kg/day; PBS/5% Tween 80 was administered to the control group. A/ phosphorylation on Tau epitope Thr231, B/ phosphorylation on Tau epitopes Ser396&Ser404 using AD2 antibody. Data are mean±SEM, n=7 animals per group. Statistical analysis is Student t-test. Significance is *P<0.05.

EXAMPLES Abbreviations

-   ANOVA—analysis of variance -   ARC—nucleus arcuatus -   GSK-3β—glycogen synthase kinase—3β -   MWM—Morris water maze -   PDK-1—phosphoinositide-dependent kinase—1 -   SC—subcutaneous -   SDS—sodium dodecyl sulfate -   SEM—standard error of the mean -   WB—Western blot -   LiPR31—an analog of PrRP31 palmitoylated at Lys¹¹ through gamma     glutamic acid linker

Tested Compounds

According to the structure-activity studies, analogs of PrRP31 and PrRP20, rat (identical to mouse) or human, lipidated at N-terminal or amino acid containing amino group, using C14 or C16 fatty acid. Methionine in position 8 in PrRP31 was replaced by more stable norleucine. In Thy Tau22 mice study, an analog of PrRP31 palmitoylated at Lys¹¹ (Lys instead of Arg) through gamma glutamic acid linker (hereinafter referred to as LiPR31) was used.

Lipidated analogs of PrRP31 and PrRP20 were synthesized by solid-phase synthesis at the IOCB AS CR, Prague, on the peptide synthesis department, as described by Maixnerová et al. (Maletínská et al., 2007).

Liraglutide was purchased from Novo Nordisk A/S (Bagsvaerd, Denmark).

Animals

To examine neuroprotective properties, insulin resistant animal model was used, e.g. mouse model with obesity induced by MSG. These mice are characterized by growth hormone insufficiency, pituitary and optic nerves atrophy, and infertility (Olney, 1969). In their brains the reduced nucleus arcuatus, enlarged third brain ventricle, and narrowed eminentia mediana are observed. Total number of neurons in ARC is reduced about 75% in MSG mice compared to their controls; however, the number of neurons does not differ significantly in other brain regions (Elefteriou et al., 2003).

The imbalance between food intake and energy expenditure at MSG obese mice leads to hypophagia and an increased adipose tissue; compared to their control, MSG obese mice have even 8 times higher weight of white adipose tissue (Maletínská et al., 2006). They have also increased leptin and glucose blood concentration, and insulin resistance (Maletínská et al., 2006).

For evaluation of the the neuroprotecitve effect of palmitoylated PrRP LiPR31, the model of AD like pathology, Thy-Tau22 mice, was used. Thy-Tau22 mice overexpress human 4R-Tau protein with mutations G272V and P3015. These mice develop memory deficits, Tau hyper-phosphorylation at different epitopes, such as Ser202, Thr205, Thr212, Ser214, Thr231, Ser396, in CA1 region of hippocampus, and neurofibrillary tangles formation (Schindowski et al., 2006; Van der Jeugd et al., 2011).

MSG Mice

Mice of strain NMRI (Harlan, Italy) were housed at the certified animal facility of IOCB AS CR, Prague, in the campus of Academy of Science in Kr{hacek over (c)} at 22±2° C., they had free access to water and food. They were fed standard chow diet St-1 (Mlýn Kocanda, Jesenice, Czech Republic), which contained 66% calories as carbohydrates, 25% as protein, and 9% as fat; its energy content was 3.4 kcal/g. Daily cycle was 12/12 hours, lights on at 6:00 a.m. All animal experiments followed the ethical guidelines for animal experiments and the Czech Republic Act No. 246/1992.

For obesity induction, the newborn NMRI mice were SC administered with sodium glutamic acid (Sigma, St. Louis, USA) at dose 4 mg/g of body weight at postnatal days 2-5. These MSG-obese mice were fed the same standard diet as the control group. The food and body weight was monitored once per week. For the study, MSG and control male mice at the age of 2 and 6 months were used.

6-Month-Old MSG Mice Treatment with Peptides Increasing Insulin Sensitivity

Groups of MSG mice (n=10 animals per group) were for 14 days SC administered with liraglutide at a dose 0.2 mg/kg, or palmitoylated analog of PrRP31 at a dose 5 mg/kg, or palmitoylated analog with PrRP31 with dichlorophenylalanin in position 31 at a dose 5 mg/kg dissolved in saline, twice a day, at 8 a.m. and 6:00 p.m. Control mice (n=10 animals per group), NMRI and MSG, were injected with saline (the volume was always 0.2 ml/mouse).

Spatial Memory Testing at 6 Months Old MSG Mice

The spatial memory was tested using Morris water maze (MWM) following the protocol described in article of Vorheese and Williamse (Vorhees and Williams, 2006) in 6-month-old MSG mice and their age-matched controls.

Thy-Tau22 Mice

Thy-Tau22 female mice and their age-matched WT controls (C57B1/6 origin) were a kind gift from INSERM laboratory, Lille, France, the research group “Alzheimer & Tauopathies”. Mice were obtained at the age of 7 months, and were housed 3-4 per cage in the certified animal facility of the Institute of Physiology AS CR, Prague, Czech Republic, with free access to water and Altromin diet (Altromin, Eastern-Westphalia, Germany). Daily cycle was 12/12 hours, lights on at 6:00 a.m. All animal experiments followed the ethical guidelines for animal experiments and the Czech Republic Act No. 246/1992.

Thy-Tau22 Mice Treatment with LiPR31

Thy-Tau22 mice were infused for 2 months with LiPR31, with doses 5 mg/kg/day dissolved in PBS/5% Tween 80 pH 6, using SC Alzet® osmotic pumps. Control mice were infused with PBS/5% Tween 80. Alzet® osmotic pumps were subcutaneously (SC) implanted in short-term ether anesthesia, and were changed after one months of experiment.

Spatial Memory Testing of Thy-Tau22 Mice

The spatial memory was tested before the beginning of the treatment with LiPR31, and after 2 months of the treatment, using the Y-maze. Experiment was performed following the protocol described by Belarbi et al. (Belarbi et al., 2011)

Tissue Dissection

Overnight fasted mice with ad libitum access to water were weighed, and their plasma glucose concentration was measured using Glucocard glucometer. After decapitation, the brains were dissected on ice, and cut between hemispheres. For immunohistochemical staining the half of the brain was fixed for 24 hours in 4% paraformaldehyde and dehydrated in 70% ethanol, afterward. For the western blot (WB) analysis, the hippocampus was dissected, and lysed in cold lysis buffer (62.5 mmol.l⁻¹ Tris-HCl, pH 6.8 with 1% sodium deoxycholate, 1% Triton X-100, Complete, 50 mmol.l⁻¹ NaF, 1 mmol.l⁻¹ Na₃VO₄), homogenized, sonicated 10 minutes and stored at −20° C. The blood plasma was prepared, and stored at −20° C.

Western Blot Analysis of Proteins Implicated in Insulin Signaling Cascade and Detection of Hyperphosphorylation of Tau Protein

In homogenized hippocampi the protein level was measured using BCA kit (Pierce, Thermo Fisher Scientific, Rockfor, Ill., USA), then the samples were diluted in sample buffer (62.5 mmol.l⁻¹ Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 0.01% bromfenol blue, 5% merkaptoethanol, 50 mmol/l NaF and 1 mmol/l Na₃VO₄) to final concentration 1 ug/μl. WB method and analysis of the results were performed according to Nagelova et al. (Nagelova et al., 2014). The list of the used antibodies and their dilution is shown in table 1.

TABLE 1 Antibody Company Dilution Rabbit monoclonal antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20 against Phospho-Akt (Ser473) Beverly, MA, USA Rabbit monoclonal antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20 against Phospho-Akt (Thr308) Beverly, MA, USA Rabbit monoclonal antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20 against total Akt Beverly, MA, USA Rabbit monoclonal antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20 against Phospho-GSK-3β (Ser9) Beverly, MA, USA Rabbit monoclonal antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20 against total GSK-3β Beverly, MA, USA Rabbit monoclonal antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20 against Phospho-PDK1 (ser241) Beverly, MA, USA Rabbit monoclonal antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20 against total PDK1 Beverly, MA, USA AD2 rabbit monoclonal Tau Gift from Dr. M.-C.Galas, 1:10 000 5% milk TBS/tween-20 antibody [pS396 & pS404] Inserm, Lille, Francie Rabbit polyclonal antibody Invitrogen Grand Island, NY, 1:10 000 5% BSA TBS/tween-20 against Tau [pS396] USA Rabbit polyclonal antibody Invitrogen Grand Island, NY, 1:1000 5% BSA TBS/tween-20 against Tau [pT231] USA Rabbit polyclonal antibody Invitrogen Grand Island, NY, 1:1000 5% BSA TBS/tween-20 against Tau [pT212] USA Anti-total Tau CTer Gift from Dr. M.-C.Galas, 1:10 000 5% milk TBS/tween-20 Inserm, Lille, Francie Anti-total Tau NTer (M19G) Gift from Dr. M.-C.Galas, 1:10 000 5% milk TBS/tween-20 Inserm, Lille, Francie Mouse monoclonal antibody Millipore, Billerica, MA, USA 1:10 000 5% milk TBS/tween-20 against Tau1 (Ser195, 198, 199, 202) Mouse monoclonal antibody Sigma, St. Louis, MO, USA 1:10 000 5% milk TBS/tween-20 against β-actin

Immunohistochemical Staining of Hyperphosphorylated Tau Protein

To verify the results obtained from WB analysis the immunohistochemical staining was performed. 10 μm thick paraffin-embedded brain slices were prepared at INSERM, Lille, France. Immunohistochemical staining was performed according the method from Violet et al. (Violet et al., 2014).

Statistical Analysis

Statistical analysis was calculated by 1-way ANOVA, with Dunnett post-hoc test, or by Student t-test, using GrapPad software (San Diego, Calif., USA). Data are presented as mean ±SEM.

Results:

MWM with 6-Month-Old MSG Obese Mice

The escape latency was measured in 6-month-old MSG mice and their age-matched controls. Experiment was performed 5 days with 4 sessions per day. As shown in FIG. 1, MSG mice had significantly increased escape latency compared to the control group.

Insulin Signaling Activation and Tau Phosphorylation in Hippocampi of 2- and 6-Month-Old MSG Mice and Their Controls

Activation of insulin signaling cascade and Tau protein phosphorylation was measured by WB analysis in hippocampi of MSG obese mice and their controls aged 2 and 6 months. The phosphorylation of GSK-3β at Ser9 was detected. As shown in FIG. 2A, the phosphorylation was decreased in MSG mice at the age of 2 months, and furthermore significantly decreased at the age of 6 months, compared to the control mice. A decreased phosphorylation of Ser9 at GSK-3β probably caused increased phosphorylation of Tau protein at epitopes Ser396 and Thr231, as shown in FIG. 2B and 2C. The 6-month old MSG mice were proven as suitable model for testing the effect of insulin-sensitizing compounds.

Insulin Signaling Cascade in Hippocampi of 6-Month-Old MSG Mice after 14-Day Treatment with Palmitoylated Analog of PrRP31 and Liraglutide

Enhanced activation of kinases implicated in insulin signaling cascade was observed in hippocampi of 6-month-old MSG mice after 14-day intervention, either with palmitoylated analog of PrRP31, or with liraglutide, as shown in FIG. 3. After liraglutide treatment, significantly increased phosphorylation was observed in PDK-1, Akt (Thr308), and GSK-3β (Ser9); more pronounced phosphorylation was observed in-Akt (Thr308), and GSK-3β (Ser9) after treatment with palmitoylated analog of PrRP31.

Tau Phosphorylation in Hippocampi of 6-Month-Old MSG Mice after 14-Day Treatment with Palmitoylated Analog or PrRP31 and Liraglutide

Tau phosphorylation in hippocampi of 6 month-old-MSG mice after 14-day treatment was measured using WB analysis. In accordance with previous results, the increased phosphorylation of GSK-3β at Ser9 caused decreased phosphorylation of Tau protein at epitopes Ser396, Thr212 and Trh231 after 14-day-long treatment either with palmitoylated analog of PrRP31, or with liraglutide, as shown in FIG. 4A, B and C. Antibody Tau1, which recognizes not-phosphorylated Tau, did not show any significant differences among groups (FIG. 4D).

Phosphorylation of GSK-3β and Tau Protein at Epitope Thr231 in Hippocampi of 6-Month-Old MSG Mice after 14-Day-Long Treatment with palmitoylated analog of PrRP31 with Dichlorophenylalanin in Position 31

Phosphorylation was detected using the method of WB. As shown in FIG. 5, 14-day-long treatment with palmitoylated analog of PrRP31 with dichlorophenylalanin in position 31 increased phosphorylation of GSK-313 at Ser9 and subsequently led to a decreased phosphorylation of Tau protein at the epitope Thr231.

Immunohistochemical Fluorescent Double Staining of Tau Phosphorylation in CA1 Region of Hippocampi of 6-Month-Old MSG Mice after 14-Day-Long Intervention with Palmitoylated Analog of Prrp31 and Liraglutide

To evaluate the WB analysis the double immunohistochemical staining was used. As shown in FIG. 6, the phosphorylation of Tau protein at epitopes Thr212 and Ser202/Thr205 was increased in MSG mice at the age of 6 months compared to their age-matched control, both treated with saline. Increased phosphorylation is manifested by a stronger fluorescent signal using the laser of the same intensity.

After 14-day treatment with palmitoylated analog of PrRP31 and liraglutide, the Tau phosphorylation is decreased in hippocampal region CA1, which is manifested by a weaker fluorescent signal, using the laser of the same intensity.

Spatial Memory Testing in Thy-Tau 22 Mice before and after the Treatment with LiPR31

The spatial memory was tested before and after the treatment with LiPR31 in Thy-Tau22 mice and their age-matched WT control using the Y-maze; the WT and Thy-Tau22 control group was treated with PBS/5% Tween 80. As shown in FIG. 7A before the experiment the Thy-Tau22 mice spent significantly less time in the newly open arm, compared to WT animals. After the 2-month-long treatment with LiPR31 the Thy-Tau22 mice spent significantly more time in the new arm compared to the PBS/Tween 80 treated group, as shown in FIG. 7B.

Tau Phosphorylation in Hippocampi of 9-month-old Thy-Tau22 Mice after 2-Month-Long Treatment with LiPR31

Tau phosphorylation was determined in the hippocampi of Thy-Tau22 mice treated with LiPR31 and their Thy-Tau22 control using the method of WB. Compared to the control group, the attenuation of Tau phosphorylation at epitopes Thr231, Ser396 and Ser404 was observed in hippocampi of Thy-Tau22 mice treated for 2 months with LiPR31, as shown in FIG. 8A and 8B.

Conclusions

AD is characterized by two pathological changes in neurons: formation of non-soluble extracellular Aβ plaques and hyperphosphorylation of intracellular cytoskeletal Tau protein.

Within the framework of the present invention, the potential neuroprotective effect of tested compound was examined in the mouse model of obesity and insulin resistance, where obesity is caused by the application of monosodium glutamate (MSG) to newborn animals. Thy-Tau 22 mice, a model of AD like pathology, were also used to verify neuroprotective effect of tested compound.

Compounds increasing insulin sensitivity were tested regarding their effect on insulin signaling cascade and tau hyperphosphorylation in the brain (in hippocampus), in MSG obese mice before and after peptides application.

Compared to age-matched controls, the insulin resistance was observed in the brain of MSG obese mice. Decreased activation of insulin signaling cascade led to a decreased phosphorylation of GSK-313 at Ser9, which increased its kinase activity. GSK-3β is one of the most important kinases implicated in Tau phosphorylation. Consequently, hyperphosphorylation of Tau protein was observed at epitopes Ser396 and Thr231. After 14-day treatment with compounds increasing insulin sensitivity, which were palmitoylated analog of PrRP31, palmitoylated analog of PrRP31 with dichlorophenylalanin in position 31, and analog of GLP-1 liraglutide which served as a positive control, an enhanced activation of insulin signaling cascade, including increased phosphorylation of GSK-3β at Ser9, and decreased phosphorylation of Tau protein at epitopes Ser396, Thr212 and Thr231 was observed.

Tested analogs of palmitoylated PrRP enhanced insulin signaling cascade in the hippocampi of 6-month-old insulin resistant MSG mice after 14-day SC treatment. Attenuated Tau phosphorylation was also observed; Tau hyperphosphorylation is the pathological change found in brains of AD patients.

INDUSTRIAL APPLICABILITY

New analogs of prolactin releasing peptide (PrRP) represent neuroprotective agents for peripheral treatment and prevention of diseases, which are Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.

REFERENCES

-   Belarbi K, Burnouf S, Fernandez-Gomez F J, Laurent C, Lestavel S,     Figeac M, Sultan A, Troquier L, Leboucher A, Caillierez R, Grosjean     M E, Demeyer D, Obriot H, Brion I, Barbot B, Galas M C, Staels B,     Humez S, Sergeant N, Schraen-Maschke S, Muhr-Tailleux A, Hamdane M,     Buee L and Blum D (2011), Neurobiol Dis 43:486-494. -   Elefteriou F, Takeda S, Liu X, Armstrong D a Karsenty G (2003),     Endocrinology 144:3842-3847. -   Hinuma S, Habata Y, Fujii R, Kawamata Y, Hosoya M, Fukusumi S,     Kitada C, Masuo Y, Asano T, Matsumoto H, Sekiguchi M, Kurokawa T,     Nishimura O, Onda H a Fujino M (1998), Nature 393:272-276. -   Maletínská L, Maixnerová J, Matysková R, Haugvicová R, Sloncová E,     Elbert T, Slaninova J a {hacek over (Z)}elezná B (2007), Eur J     Pharmacol 559:109-114. -   Maletínská L, Toma R S, Pirnik Z, Kiss A, Slaninová J, Haluzik M a     {hacek over (Z)}elezná B (2006), Regul Pept 136:58-63. -   Nagelová V, Pirnik Z, {hacek over (Z)}elezná B a Maletínská L     (2014), Brain Res 1547:16-24. -   Olney J W (1969), Science 164:719-721. -   Schindowski K, Bretteville A, Leroy K, Begard S, Brion J P, Hamdane     M and Buee L (2006), Am J Pathol 169:599-616. -   Van der Jeugd A, Ahmed T, Burnouf S, Belarbi K, Hamdame M, Grosjean     M E, Humez S, Balschun D, Blum D, Buee L and D'Hooge R (2011),     Neurobiology of learning and memory 95:296-304. -   Violet M, Delattre L, Tardivel M, Sultan A, Chauderlier A,     Caillierez R, Talahari S, Nesslany F, Lefebvre B, Bonnefoy E, Buee L     a Galas M C (2014), Frontiers in cellular neuroscience 8:84.

Vorhees C V a Williams M T (2006), Nature protocols 1:848-858. 

1. Lipidated neuropeptides based on prolactin-releasing peptide, selected from prolactin-releasing peptide 20 (PrRP20), prolactin-releasing peptide 31 (PrRP31) and their analogs, wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; said PrRP-based neuropeptide containing C14 and/or C16 fatty acid chain, said fatty acid is bound in position 1 or 11 for PrRP31 or its analogs and in position 1 or 7 for PrRP20 or its analogs; said fatty acid being bound by a bond between an amino acid having at least one free NH₂, OH or SH group and the carboxylic group of the fatty acid or through a hydrophilic linker X² selected from the group comprising polyoxyethylene moiety, arylalkyl moiety, or a saturated or unsaturated, linear or branched C₃-C8 hydrocarbon chain, wherein some carbon atoms may be replaced by heteroatoms selected from a group comprising N, S, and O; said chain carrying at least one and preferably two amino groups or carboxylic acid groups, one of which may be substituted to form a group selected from: CONH₂; NH-polyoxyethylene; COOM¹ wherein M¹ is alkali metal, preferably Na or K; CN; COOR¹, COR¹, or CONHR¹ wherein R¹ is selected from a group comprising lower alkyl, arylalkyl, polyoxyethylene, methylpolyoxyethylene, and aminoethylpolyoxyethylene; (CHOH)_(n)R² wherein R² is H or COOH and n is an integer from 2 to 10; or (CH)_(n)N⁺R₃, wherein R³ is the same or different, selected from H and C₁-C₄ alkyl; and the PrRP31 or its analogs may optionally have the amino acid in position 11 replaced by an amino acid having a free NH₂, OH or SH group, particularly when the fatty acid is bound in position 11 for PrRP31 or its analogs; and the PrRP20 or its analogs may optionally have the amino acid in position 7 replaced by an amino acid having a free NH₂, OH or SH group in position 7, particularly when the fatty acid is bound in position 7 for PrRP20 or its analogs; for use in a method of treatment and prevention of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.
 2. Lipidated neuropeptides according to claim 1, wherein isoleucine can be replaced by phenylglycine or alanine, valine can be replaced by phenylglycine and/or terminal phenylalanine can be replaced by dichlorophenylalanine, pentafluorophenylalanine, nitrophenyalanine, histidine, benzylhistidine, naphtylalanine, tryptofane, pyroglutamic acid, benzylcysteine, benzyl-O-glutamate, tetrachlorophenylalanine, methyl-O-phenylalanine or methyl-NH-phenylalanine, in the sequence of the C-terminal heptapeptide.
 3. Lipidated neuropeptides according to claim 1, wherein X² is a hydrophilic linker selected from the group comprising β-alanine, γ-aminobutyric acid and γ-glutamic acid.
 4. Lipidated neuropeptides according to claim 1 having general formulae selected from: (1) (X)SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH₂, (2) (X)SRAHQHSMETRTPDINPAWYTGRGIRPVGRF-NH₂, (3) (X)TPDINPAWYASRGIRPVGRF-NH₂ (4) (X)TPDINPAWYTGRGIRPVGRF-NH₂,

wherein X═X¹ or X¹X²; X¹ being tetradecanoic or hexadecanoic acid, which is bound in a position 1 to an amino acid of the above mentioned peptide chain either directly or through X², X² being a hydrophilic linker as defined in claim 1, preferably selected from the group consisting of β-alanine, □-amino butyric acid and □-glutamic acid, and wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; for use in the treatment and prevention, preferably by peripheral administration, of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.
 5. Lipidated neuropeptides according to the claim 1 having formulae selected from: (5) (palm)SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH₂ and (6) (palm)TPDINPKWYASRGIRPVGRF-NH₂;

wherein palm is hexadecanoic acid, and wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; for use in the treatment and prevention, preferably by peripheral administration, of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders.
 6. Lipidated analogs according to claim 1 having formulae selected from: (7) SRTHRHSMEIK(palm)TPDINPAWYASRGIRPVGRF-NH₂, (8) TPDINPK(palm)WYASRGIRPVGRF-NH₂, (9) SRTHRHSMEIKTPDINPAWYASRGIRPVGRF-NH₂,  and           |            X²(palm) (10) TPDINPKWYASRGIRPVGRF-NH₂;       |      X²(palm)

wherein palm is hexadecanoic acid and X² is □-glutamic acid, and wherein in the C-terminal sequence IRPVGRF-NH₂, one or more of isoleucine, valine and phenylalanine can be replaced by another amino acid; for use in the treatment and prevention, preferably by peripheral administration, of diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), cognitive impairment no dementia (CIND), brain trauma, and neurodegenerative changes and disorders. 